The rate of membrane receptor trafficking is critical for the steady-state level of surface expression and is tightly coupled to signaling events (1-3). Within a given molecule, such as K+ channels, various trafficking sequence motifs have been identified that contribute to the specificity of trafficking behavior (4). For a given receptor, recent evidence suggests that the rate of trafficking may change according to the physiological state, such as during cellular aging (5). To assess the transit time from the endoplasmic reticulum (ER) to the cell surface, a conventional approach is to measure the time of ER-to-Golgi transition, which is thought to be the rate-limiting step. The remaining steps of biogenesis usually take a very short time, ranging from seconds to minutes (6). The ER-to-Golgi transition time usually is determined by pulse-chase labeling combined with monitoring a shift of molecular weight as a result of glycosylation. This technique has been particularly useful for studies to determine specific organelle transitions, such as a rate change from ER to Golgi by forward transport signals (7). However, for proteins with limited or no glycosylation, this approach is not applicable. Furthermore, many receptors and ion channels undergo a stationary step in their trafficking cascade after exiting from trans-Golgi and before cell-surface expression. For example, only a fraction of synthesized nicotinic acetylcholine receptor matures and expresses on the cell surface (8). The combination of different transport rates, maturation pathways, and posttranslational modifications warrants a more in-depth consideration of methods to directly monitor the rate by which a given receptor complex populates and/or repopulates the cell surface after exiting from the Golgi apparatus.
In addition to the ER-Golgi trafficking, one critical spatial transition underlying diverse biological activity is the regulation of protein expression on the cell surface (for review, see ref. 9). Fluorescence recovery after photobleaching (FRAP) was developed for imaging the movement of biological molecules in cells (10, 11). This method has been improved greatly with extensive use of genetically coded fluorophores, such as green fluorescent protein (GFP), providing a powerful means to address questions regarding protein localization, activity, interactions, and dynamics with living cells (12). This approach has high spatial resolution at the single-cell level. However, the combination of optic detection and the photobleaching methodology limits its applicability in certain areas, especially evaluation of global surface expression and coupling the imaging signal to functionality of targeted molecules.
There is a continuing need in the art to develop methods for assaying cell surface protein transit. The assays should be useful whether or not a protein is glycosylated. The assays should be suitable for distinguishing cell surface molecules in different states, including but not limited to distinguishing functional from non-functional cell surface molecules.